High iron levels in blood and tissues are a risk factor for diabetes mellitus. This is true not only for well- described conditions of iron overload such as hereditary hemochromatosis (HH) but even for dietary iron excess resulting in serum ferritin concentrations within the higher ranges of normal. In our previous funding cycle we determined that serum ferritin, as a measure of tissue iron levels and not as a reflection of inflammation is elevated in diabetes and metabolic syndrome. Ferritin also correlates inversely with levels of adiponectin, the insulin sensitizing adipokine. We established in mice and humans a causal role for iron in decreasing insulin secretion and adiponectin: We achieved significant protection from diabetes by dietary iron restriction or chelation in the Obese mouse, and replicated the findings in humans in a pilot study of the effects of phlebotomy in pre-diabetes (impaired glucose tolerance). In the course of this work, other important and heretofore unknown connections between dietary iron and metabolism emerged. These include: (1) Glucose regulation of hepcidin, the main regulator of systemic iron absorption and disposition, and (2) variation of circadian metabolic rhythmicity with dietary iron. These pathways provide novel links between iron and the known diabetes risk factors of inflammation and disturbed circadian rhythm. We propose to study the mechanisms for these associations in animal models because it is crucial to understand the ramifications of altering body iron stores to ameliorate diabetes risk. Our Specific Aims: 1. Determine the mechanism of the regulation of hepcidin by glucose. Hepcidin is a small (25 a.a.) peptide produced by the liver in response to rising serum iron levels. It binds to and down regulates ferroportin, an iron channel through which dietary iron is released from the duodenum into the circulation. This creates a negative feedback loop for systemic iron absorption. Beyond its effects on iron absorption, however, hepcidin functions as an innate immune molecule. Its production is stimulated by inflammatory cytokines and hepcidin itself is anti-inflammatory. We found that glucose administration to mice and humans induces a serum activity that leads to a rapid and profound decrease in hepcidin activity. We propose to complete the purification of this activity, and determine its source and regulation. 2. Determine the consequences of the regulation of hepcidin by glucose on systemic iron balance and inflammatory stress. We show that the acute down regulation of hepcidin by glucose allows increased iron absorption, but the consequences of chronic regulation are not known. We hypothesize that this regulation, and the dysregulation that we demonstrate occurs in diabetes, should have important consequences for iron homeostasis and inflammation, both of which have been demonstrated to contribute to diabetes pathogenesis. The effects of glucose on hepcidin, and subsequent effects on inflammatory signaling in macrophages and adipocytes, will be examined in mouse models with normal glucose tolerance, hyperphagia, diabetes, and with genetic inactivation of the hepcidin gene. 3. Determine the consequences of dietary iron levels within the normal range on circadian metabolic rhythmicity, and explore the mechanisms for that regulation. Our preliminary data demonstrate that modest changes in levels of dietary iron affect circadian regulation of metabolism, in particular of hepatic glucose production. We hypothesize that these effects may provide a mechanism that links the risks for diabetes engendered by altered sleep/wake cycles with those of excess dietary iron. We will therefore explore circadian regulation of hepatic glucose production in mice fed dietary iron within the low and high ranges of normal, wherein neither anemia nor overt iron overloads result. Initial exploration of mechanisms will include altered heme and NAD levels, the latter resulting in altered AMPK signaling. PUBLIC HEALTH RELEVANCE: A large body of evidence now points to a strong relationship between body iron stores and diabetes risk, and diabetes is a major problem especially in the aging veteran population. Our published and preliminary data on the effects of a low iron diet in mouse models of type 2 diabetes, and of lowering iron by blood donation in humans with impaired glucose tolerance, open new pathways toward treatments of type 2 diabetes. Most of the mechanisms and details underlying the relationship of iron to diabetes risk, however, remain unknown, and it is crucial to understand the basis for these relationships as we set out to reverse them. This proposal is to better understand the relationship of iron to diabetes. We discovered that metabolism affects the key regulator of iron balance, and we will study the mechanism for this. We will also test the effects of dietary iron on the circadian clock, because it is known that its disruption can have major effects of diabetes risk.